Tryptophan Scanning Mutagenesis in the TM3 Domain of the Torpedo californica Acetylcholine Receptor Beta Subunit Reveals an α-Helical Structure

We used tryptophan substitutions to characterize the beta M3 transmembrane domain (βTM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the βTM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and e...

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Bibliographic Details
Published in:	Biochemistry (Easton) Vol. 43; no. 31; pp. 10064 - 10070
Main Authors:	Santiago, John, Guzmán, Gisila R, Torruellas, Karla, Rojas, Legier V, Lasalde-Dominicci, José A
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 10-08-2004
Subjects:	Acetylcholine - pharmacology Amino Acid Substitution - genetics Animals Binding Sites - drug effects Binding Sites - genetics Dose-Response Relationship, Drug Marine Mutagenesis, Site-Directed Oocytes - metabolism Patch-Clamp Techniques Protein Binding - drug effects Protein Binding - genetics Protein Structure, Secondary - genetics Protein Structure, Tertiary - genetics Protein Subunits - biosynthesis Protein Subunits - chemistry Protein Subunits - genetics Receptors, Cholinergic - biosynthesis Receptors, Cholinergic - chemistry Receptors, Cholinergic - genetics Torpedo Torpedo californica Tryptophan - genetics Xenopus Xenopus laevis USA, Washington
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Summary:	We used tryptophan substitutions to characterize the beta M3 transmembrane domain (βTM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the βTM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [125I]-α-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282−296 of the βTM3 domain was consistent with an α-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed α-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica β subunit.
Bibliography:	istex:F58EB8596707B4B2E9C3AFD84DF58842A96ACED9 This research was supported by National Institutes of Health Grants 2GM56371-05 GM08102-27 and by RCMI-G12RR03035-16. John Santiago was supported by the Puerto Rico NSF-EPSCoR Grant EPS-97478, and Gisila Guzmán by the MBRS−RISE Program 5R25GM61151. ark:/67375/TPS-25FS6JPS-1 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0006-2960 1520-4995
DOI:	10.1021/bi0362368